Sandra D. Mulder

Astrocytic A beta 1-42 Uptake Is Determined by A beta-Aggregation State and the Presence of Amyloid-Associated Proteins

Intracerebral accumulation of amyloid‐β (Aβ) leading to Aβ plaque formation, is the main hallmark of Alzheimers disease and might be caused by defective Aβ‐clearance. We previously found primary human astrocytes and microglia able to bind and ingest Aβ1‐42 in vitro, which appeared to be limited by Aβ1‐42 fibril formation. We now confirm that astrocytic Aβ‐uptake depends on size and/or composition of Aβ‐aggregates as astrocytes preferably take up oligomeric Aβ over fibrillar Aβ. Upon exposure to either fluorescence‐labelled Aβ1‐42 oligomers (Aβoligo) or fibrils (Aβfib), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Aβ‐internalization was verified using confocal microscopy and live‐cell imaging. Neither uptake of Aβoligo nor Aβfib, triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin‐6 and monocyte‐chemoattractant protein‐1 release. Amyloid‐associated proteins, including α1‐antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Aβ‐aggregation. Here, astrocytic uptake of Aβfib increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Aβoligo‐positive astrocytes, whereas SAP/C1q slightly reduced Aβoligo uptake. Thus, amyloid‐associated proteins, especially ApoJ and ApoE, can alter Aβ‐uptake in vitro and hence may influence Aβ clearance and plaque formation in vivo.

The effect of amyloid associated proteins on the expression of genes involved in amyloid-beta clearance by adult human astrocytes

Astrocytes appear to be important mediators in the clearance of amyloid beta1-42 (Aβ), the key component of senile plaques characteristic of Alzheimers disease (AD). Recently, we found the amyloid associated proteins (AAPs) α1-antichymotrypsin (ACT), apolipoprotein J and E (ApoJ and ApoE) and a mixture of serum amyloid P (SAP) and C1q (SAP-C1q) to modify Aβ-uptake by human astrocytes. Here we investigated the effect of oligomeric (Aβoligo) and fibrillar Aβ (Aβfib), alone and in combination with a panel of AAPs on the astrocytic expression of genes proposed to be involved in Aβ-uptake and degradation. Primary human astrocytes (isolated from non-demented control (n=4) and AD patient (n=4) brain specimens) were exposed to either Aβoligo or Aβfib preparations with or without the above mentioned AAPs. Quantitative gene expression analysis of Aβ-receptors Scavenger receptor B1 (SCARB1), macrophage receptor with collagenous structure (MARCO) and low density lipoprotein receptor related protein-2 (LRP2 or megalin) as well as of Aβ-degrading enzymes neprilysin (NEP), insulin-degrading enzyme (IDE) and metalloproteinase-9 (MMP-9) was performed by real-time PCR. Basal expression of NEP, IDE and SCARB1 was easily detected whereas expression of MARCO, LRP2 and MMP-9 could only be detected upon pre-amplification. Basal expression of NEP, IDE and SCARB1 did not change upon exposure to Aβoligo or Aβfib alone in any of the investigated astrocyte cultures. Interestingly NEP expression was increased upon exposure to ApoE in combination with both Aβ-preparations, and also SCARB1 expression was induced upon treatment with ApoE in combination with Aβfib in astrocytes from non-demented controls. Further, SAP-C1q increased SCARB1 expression in control astrocytes when combined with Aβoligo. These alterations were not found in astrocytes from AD patients. Thus, we conclude that Aβ alone apparently does not affect the astrocytic expression of IDE, NEP or SCARB1. However, NEP and SCARB1 expression is increased in astrocytes from non-demented subjects when exposed to Aβ combined with AAPs like ApoE. These astrocytic gene expression-regulatory mechanisms appear to be defective in AD and thus might contribute to the development and progression of AD pathology.

BACE1 Activity in Cerebrospinal Fluid and Its Relation to Markers of AD Pathology

Several studies have shown that reduced amyloid-beta 1-42 (Abeta(42)) and increased tau levels in cerebrospinal fluid (CSF) reflect increased Alzheimers disease (AD) pathology in the brain. beta-site APP cleaving enzyme (BACE1) is thought to be the major beta-secretase involved in Abeta production in the brain, and therefore we investigated the relation between BACE1 activity and CSF markers Abeta(40), Abeta(42), total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau) in CSF of control (n=12), mild cognitive impairment (n=18), and AD (n=17) subjects. Patients were classified according to their Abeta(42), t-tau, and p-tau CSF biomarker levels, with either an AD-like biomarker profile (two or three biomarkers abnormal: Abeta(42) < 495 pg/ml in combination with t-tau > 356 pg/ml, and/or p-tau > 54 pg/ml) or a normal biomarker profile (<or= one biomarker abnormal). This resulted in 19 subjects with an AD-like biomarker profile (66 +/- 6 years, 53% female, and Mini-Mental Status Examination (MMSE) score: 23 +/- 5) and 28 subjects with a normal biomarker profile (62 +/- 11 years, 43% female, and MMSE score: 27 +/- 4). Subjects with an AD-like biomarker profile had higher CSF BACE1 activity levels, compared to patients with a normal biomarker profile (20 pg/ml and 16 pg/ml respectively; p=0.01), when controlled for age and gender. In the whole sample, BACE1 activity correlated with CSF levels of Abeta(40), t-tau, and p-tau (r=0.38, r=0.63, and r=0.65; all p< 0.05), but not with Abeta(42). These data suggest that increased BACE1 activity in CSF relates to AD pathology in the brain.

Apolipoproteins E and J interfere with amyloid-beta uptake by primary human astrocytes and microglia in vitro.

Defective clearance of the amyloid‐β peptide (Aβ) from the brain is considered a strong promoter in Alzheimers disease (AD) pathogenesis. Astrocytes and microglia are important mediators of Aβ clearance and Aβ aggregation state and the presence of amyloid associated proteins (AAPs), such as Apolipoproteins E and J (ApoE and ApoJ), may influence Aβ clearance by these cells. Here we set out to investigate whether astrocytes and microglia differ in uptake efficiency of Aβ oligomers (Aβoligo) and Aβ fibrils (Aβfib), and whether the Aβ aggregation state and/or presence of AAPs affect Aβ uptake in these cells in vitro. Adult human primary microglia and astrocytes, isolated from short delay post‐mortem brain tissue, were exposed to either Aβoligo or Aβfib alone or combined with a panel of certain AAPs whereafter Aβ‐positive cells were quantified using flow cytometry. Upon exposure to Aβ combined with ApoE, ApoJ, α1‐antichymotrypsin (ACT) and a combination of serum amyloid P and complement C1q (SAP‐C1q), a clear reduction in astrocytic but not microglial Aβoligo uptake, was observed. In contrast, Aβfib uptake was strongly reduced in the presence of AAPs in microglia, but not in astrocytes. These data provide the first evidence of distinct roles of microglia and astrocytes in Aβ clearance. More importantly we show that Aβ clearance by glial cells is negatively affected by AAPs like ApoE and ApoJ. Thus, targeting the association of Aβ with AAPs, such as ApoE and ApoJ, could serve as a therapeutic strategy to increase Aβ clearance by glial cells. GLIA 2014;62:493–503

Serum amyloid p component as a biomarker in mild cognitive impairment and Alzheimer's disease.

Background: Serum amyloid P component (SAP), present in amyloid-β (Aβ) plaques in Alzheimer’s disease (AD), may protect Aβ deposits against proteolysis, thereby promoting plaque formation. The aim was to investigate if SAP levels in cerebrospinal fluid (CSF) and serum can be used to discriminate controls, AD and mild cognitive impairment (MCI) patients, and to identify incipient AD among MCI patients. Methods: SAP levels in CSF and serum were determined in 30 controls, 67 MCI and 144 AD patients. At follow-up, 39 MCI patients had progressed to dementia, while 25 had remained stable (mean follow-up time: 2.6 ± 1.0 and 2.1 ± 0.8 years). Results: Cross-sectionally no differences were found in SAP levels in CSF and serum between the groups. MCI patients that had progressed to dementia at follow-up had lower CSF SAP levels (13 mg/l, range 3.3–199.3 mg/l) than MCI nonprogressors (20.2 mg/l, range 7.0–127.7 mg/l; p < 0.05). A low CSF SAP level was associated with a 2-fold increased risk of progression to AD (hazard ratio = 2.2; 95% confidence interval = 0.9–5.4). Conclusion: Our data suggest that measurement of CSF SAP levels can aid in the identification of incipient AD among MCI patients.

Clusterin Levels in Plasma Predict Cognitive Decline and Progression to Alzheimer’s Disease

BACKGROUND Increased clusterin levels have been reported in brain, cerebrospinal fluid (CSF), and plasma of Alzheimers disease (AD) patients. Because changes are also observed in mild cognitive impairment (MCI), a possible relationship between clusterin levels and early neurodegenerative changes in AD was suggested. OBJECTIVES To determine whether clusterin concentrations could 1) serve as a diagnostic marker for AD, 2) predict disease progression in MCI, and 3) correlate with AD-biomarkers. METHODS Clusterin levels in CSF and plasma, as well as AD biomarker levels of Aβ42, Tau, and pTau in CSF and Mini-Mental State Examination scores (MMSE) were determined in 67 controls, 50 MCI, and 107 AD patients. Repeated MMSE was obtained for 44 MCI and 72 AD patients after, on average, 2.7 years. RESULTS Elevated clusterin concentrations in plasma, but not in CSF, were a risk factor for AD (HR 18.6; 95% CI 2.8-122), and related to cognitive decline in MCI (r =-0.38; p <  0.01). An inverse relation between plasma clusterin levels and cognitive decline was observed in AD patients (r = 0.23; p≤0.05). In CSF, but not in plasma, clusterin levels correlated with Tau and pTau in all groups. CONCLUSION Elevated plasma clusterin levels in MCI confer an increased risk for progression to AD, and more rapid cognitive decline. We speculate that clusterin levels in CSF may reflect its involvement in the earliest neurodegenerative processes associated with AD pathology. Whereas neither clusterin levels in CSF nor in plasma had diagnostic value, plasma clusterin levels may serve as a prognostic marker for AD.

Evaluation of Intrathecal Serum Amyloid P (SAP) and C-Reactive Protein (CRP) Synthesis in Alzheimer's Disease with the Use of Index Values

Serum amyloid P (SAP) and C-reactive protein (CRP) are proteins involved in innate immunity. The expression of SAP and CRP is increased in Alzheimers disease (AD) brain tissue, compared to healthy controls. Although both proteins are found in cerebrospinal fluid (CSF), their origin is unclear. We investigated if increased local production of SAP and CRP in AD brain results in higher levels in CSF with the use of index values. To study this, SAP, CRP, and albumin levels were determined in CSF and serum samples of 30 control (65 ± 11 years; 57% female) and 140 AD subjects (65 ± 9 years; 53% female). To correct for inter-individual differences in protein diffusion from blood to CSF, quotients (Q =CSF/serum) of SAP, CRP, and albumin and index values (Qprotein/Qalb) were calculated. The results showed no significant differences in SAP and CRP index values between control and AD subjects, although eight percent of individual AD patients showed evidence of intrathecal SAP or CRP production using the Reiber hyperbolic model. Interestingly, the SAP index value was much lower than expected, based on its molecular size. In conclusion, these data suggest that local production of SAP and CRP in the AD brain does not substantially contribute to the CSF levels.

CSF levels of PSA and PSA-ACT complexes in Alzheimer's disease

Background Prostate-specific antigen (PSA) is a serine protease that in serum, is predominantly found complexed to the serine protease inhibitor alpha1-antichymotrypsin (ACT). ACT co-localizes with amyloid plaques in Alzheimers disease (AD) brain and both PSA and ACT are detectable in cerebrospinal fluid (CSF). Therefore, we aimed to determine whether PSA is produced in the brain and whether PSA and PSA–ACT complex levels in CSF can be used as a biomarker for AD. Methods Levels of ACT and PSA–ACT were determined by sandwich enzyme-linked immunosorbent assay in CSF and serum samples of AD (n = 16), frontotemporal lobe dementia (FTLD) (n = 19), mild cognitively impaired (MCI) patients (n = 19) and controls (n = 12). Total PSA was determined in a non-competitive immunoassay. Reverse transcriptase–polymerase chain reaction (RT–PCR) for PSA was performed on postmortem hippocampus and temporal cortex specimens from control and AD cases. Results PSA is expressed in the brain, as detected by RT–PCR. PSA and PSA–ACT complexes were detectable in CSF of almost all male and only very few female subjects. The levels of PSA and PSA–ACT complexes in CSF did not differ between AD, FTLD, MCI and control groups. PSA CSF/serum quotients highly correlated with albumin CSF/serum quotients. Furthermore, the hydrodynamic radius of PSA was found to be 3 nm and the theoretical PSA quotient, derived from the Felgenhauer plot, corresponded well with the measured PSA quotient. Conclusions PSA is locally produced in the human brain; however, brain PSA hardly contributes to the CSF levels of PSA. PSA and PSA–ACT levels in CSF are not suitable as a biomarker for AD.

Upward drift in cerebrospinal fluid amyloid β 42 assay values for more than 10 years

The best‐established cerebrospinal fluid (CSF) biomarkers for Alzheimers disease are levels of amyloid β 42 (Aβ42), total tau (tau), and phosphorylated tau 181 (ptau). We examined whether a widely used commercial immunoassay for CSF Aβ42, tau, and ptau provided stable measurements for more than 10 years.

Increased IRAK-4 Kinase Activity in Alzheimer's Disease; IRAK-1/4 Inhibitor I Prevents Pro-inflammatory Cytokine Secretion but not the Uptake of Amyloid Beta by Primary Human Glia

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β (Aβ), which is associated with a neuroinflammatory response involving microglia and astrocytes. This neuroinflammatory response has detrimental effects on disease progression but also has a beneficial function on removal of excess Aβ. Microglia and astrocytes are involved in the clearance of Aβ from the brain, but neuroinflammation also promotes neurodegeneration. In order to identify signal transduction pathways critically involved in AD we analysed human brain tissue using protein kinase activity profiling. We identified increased activity of the Interleukin 1 Receptor Associated Kinase 4 (IRAK-4) in AD compared to control brain tissue. IRAK-4 is a component of the signal transduction pathway that functions downstream of the Toll-like receptors and the interleukin-1 receptor. Immunohistochemical analysis of human brain tissue revealed the presence of IRAK-4 in astrocytes and microglia. Quantification of IRAK-4 and the phosphorylated form of IRAK-1, a specific substrate for IRAK-4, revealed increased expression and activity of IRAK-4 in AD. Interestingly, IRAK-1/4 inhibitor I reduces the lipopolysaccharide-induced secretion of monocyte chemotactic protein-1 (MCP-1) by primary human microglia and the interleukin-1β-induced secretion of MCP-1 and interleukin 6 by primary human astrocytes. In contrast, the uptake of Aβ by astrocytes and microglia is not affected by IRAK-1/4 inhibition. Our data show that IRAK-4 protein kinase activity is increased in AD and selective inhibition of IRAK-1/4 inhibits a pro-inflammatory response without affecting the uptake of Aβ by glial cells, indicating that the IRAK signalling pathway is a potential target for modulating neuroinflammation in AD.